1. Field of the Invention
The present invention relates to a method of designing a probe set for identifying a specific target sequence group by a hybridization reaction, a microarray having a substrate on which a probe designed by the method is immobilized, and a computer readable medium on which a program for executing the method is recorded.
2. Description of the Related Art
A polynucleotide microarray is a microarray having a substrate on which a polynucleotide group is immobilized with high density. The polynucleotide group is immobilized on a prescribed area of the substrate. Examples of polynucleotide microarrays are disclosed in U.S. Pat. Nos. 5,445,934 and 5,744,305.
Generally, a photolithography method has been used to manufacture a polynucleotide microarray. In this method, a polynucleotide array is manufactured by repeatedly exposing prescribed areas of a substrate where monomers protected by a removable group have been applied to energy source to remove the protective group, and repeatedly coupling the monomers protected by the removable group. Thus, polynucleotides immobilized on a polynucleotide microarray are synthesized by extending monomers one-by-one.
On the other hand, in the case of using a spotting method, a microarray is formed by immobilizing previously synthesized polynucleotides on prescribed areas. Methods of manufacturing such a polynucleotide microarray are disclosed in U.S. Pat. Nos. 5,744,305, 5,143,854, and 5,424,186, disclosures of which are included herein by reference.
Polynucleotides (referred to as a probe, a nucleic acid probe, or a polynucleotide probe) immobilized on a microarray are used to detect or identify target sequences due to the ability to hybridize to target sequences. With respect to conventional DNA probes, a standard condition for selecting a DNA probe for a target sequence is set up, and then a DNA sequence meeting the standard conditions is selected. The standard conditions or others for the selected DNA sequence are inspected to select the most preferable probe sequence. The standard conditions may include a length of a probe, Tm of a probe (the temperature at which 50% of double stranded DNA molecules are dissociated into two single stranded DNAs), a threshold value of the sequence homology with other sequences, and the like. After a candidate DNA probe meeting the standard conditions has been selected, the candidate DNA probe is examined with regard to Tm and sequence homology to determine whether or not the candidate DNA probe is unique only to a target sequence and whether or not the candidate DNA probe is a sequence that can easily induce cross hybridization. As a result, the most preferable probe among the candidate DNA probes meeting the standard conditions is selected as a sequence specifically binding to a target DNA sequence. However, in such a method of designing a probe, since a specific sequence hybridizing only to each target sequence with no cross hybridization to other sequences is selected as a probe, it is difficult to design a specific probe when sequence homology between target sequences is high.
There have been several attempts to solve the above-described problem. For example, US Patent Publication No. 2003/0069701 discloses a biochip having a substrate on which a plurality of probes for one target sequence is spotted. This disclosure relates to a method of providing additional information on a target sequence by the combination of previously inspected probes. However, the method cannot be used to design by identifying target sequences. US Patent Publication No. 2002/0160401 also discloses a biochip having a substrate on which a plurality of probes is spotted for one target sequence. This disclosure relates to a method of using information on a common probe as additional information, and a method of identifying a target sequence to design a unique probe is not disclosed.
Therefore, a method of designing a probe for identifying a target sequence from a plurality of target sequence groups by a hybridization reaction and then estimating a target sequence by the relationship between the designed probes still is required.